A powerful approach to enhance our understanding of complex biomolecular systems is to build integrated biological processes from their fundamental components. The long-term vision ofthis project is to construct a useful synthetic entity that self-replicates from small molecule building blocks and reconstitutes the macromolecular catalysts synthesizing DNA, RNA and protein. This objective demands in vitro methods for production and self-assembly of active component parts. As a tractable milestone towards this long-term vision, the current proposal will construct highly active synthetic Escherichia coli ribosomes. This project is designed to build upon the work the candidate has pursued as a K99 postdoctoral fellow under the guidance of George Church at the Harvard Medical School. Equipped with new expertise in nucleic acid biochemistry, molecular biology, and purified in vitro translation systems, this proposal takes advantage of key breakthroughs in our ability to reconstitute ribosomes under physiological conditions and enable rRNA synthesis, ribosome self-assembly, and protein synthesis in a single reaction. Specific aims will: 1) assess the efficiency of ribosome reconstitution and proportion of active synthetic ribosomes made with new selfassembly methods, 2) combine gene construction with selection and sequencing to isolate synthetic 23S rRNA demonstrating high activity in E. coli ribosomes, and 3) test the effect of in vitro variants of ribosomal component parts on ribosome function. The specific aims will add new knowledge regarding the importance of 23S rRNA sequence on ribosome activity and our mechanistic understanding of ribosomes, yield insight into evolutionary diversity of 23S rRNA sequences, impact our ability to control protein synthesis and cellular function, and enable methods to design/evolve ribosomes for understanding structure-function relationships and exploiting ribosome function. Looking forward, this work will catalyze a new paradigm for controlling and studying life, for identifying new antibiotic targets to address rising bacterial resistance, for diagnosing disease, and for greater flexibility in developing and producing effective disease treatments.